The primary objective of this proposal is to define, through in vitro and in vivo studies, the various factors important in developing protocols for therapeutic utilization of monoclonal antibodies (McAbs) directed toward human tumor associated cell surface antigens. We will utilize both McAbs and immunotoxins which are prepared by covalently conjugating McAbs with ricin A chain. As the tumor model, human leukemic-lymphoma (HLL) will be used. Thus, the in vivo studies will be carried out using HLL cells transplanted in irradiated or splenectomized-irradiated nude mice. We hope that these studies may lead to means by which we can circumvent or overcome some critical barriers in the therapeutic use of McAbs. Our approach to the generation of anti-HLL McAbs differs from the conventional one in that we are using isolated HLL cell membrane antigen preparations rather than whole cells to immunize mice in order to obtain the appropriate McAbs. The generated hybridomas are being characterized by means of a radioimmunoassay, an immunofluorescence-staining test and/or an immunoperoxidase-staining test using various human cells and tissues as the targets. By this approach we have recently generated and cloned several mouse hybridomas that produce anti-HLL McAbs. Some of these antibodies showed strikingly high specificity for HLL and appear to define unique HLL associated cell surface antigens. Further, colony-forming unit (CFU) assays are being carried out to confirm the absence of reactivity of these McAbs with hematopoietic stem cells. We will place our emphasis on killing of HLL cells by multiple-targeting, i.e., by combined use of multiple McAbs or of immunotoxins containing multiple McAbs. In order to develop protocols for the therapeutic utilization of these McAbs and immunotoxins we will carry out the following studies: (1) detailed studies of the specificity of our generated McAbs, (2) comparison of McAbs of different isotypes and IgG subclasses with respect to the efficacy of specific killing of HLL cells in vivo, (3) comparison of the intact IgG antibodies and their univalent antigen-binding derivatives with respect to their specific cytotoxic activity against HLL cells in vivo, (4) establishment of the experimental design for using McAbs and immunotoxins in the in vitro eradication of tumor cells in the bone marrow from HLL patients, and (5) investigation of various factors important for in vivo therapeutic utilization of immunotoxins.